Unit 5

5a, 5b,

Dolly the sheep experiment

1996: proved that DNA from an adult cell are sufficient to create an entire animal

Starved donor cells from a sheeps mammary gland so that it was basically all DNA left and then they fused it to an enucleated egg cell, fertilized it and it became an implanted embryo into a surrogate mother.

How is gene expression regulated?

Differentially regulated

Major classes of genes

Housekeeping genes and cell type specific genes

Housekeeping genes

Necessary for basic cell function. Ex: RNA polymerase

Cell type specific genes

Necessary for specific function. Ex: insulin for pancreatic cells

How proteins recognize dna sequences

• Non-covalent bonds form between aa side chains and base pairs in major groove

• not a strict 1-1 relationship unlike base pairing

• A given dna binding protein recognizes a variety of sequences with high affinity

• Consensus sequences

Operon

Bacterial genes arranged in clusters of related genes

Polycistronic RNA

An rna containing the whole Oberon as a single rna, encoding multiple different proteins

Promotor

Binding site for rna polymerase II and initiation factors

• Has Tata box and start site (bipartite)

Operator

DNA region with binding sites for transcriptional control proteins (activators and repressions)

Prokaryotic transcriptional unit

Prokaryotic transcriptional control: positive regulation

bound activator promotes transcription

Lac Operon

Inducible operon

Lac Z -encodes b-galactosidase: breaks lactose (a b-galactoside) into glucose and galactose

Lac Y- encodes b-galactoside permease: brings lactose into cells

Lac A- encodes b-galactoside transacetylase: transfers an acetyl group onto lactose

LacI- encodes Lac Repressor: regulates expression of Lac Operon

logic for this: bacteria dont need to make all the enzymes if lactose isn’t present in the environment

How Lac repressor binds

as a tetramer and binds to the operator, repressing transcription of LacZYA RNA

when lactose is present it binds to the lac repressors and inactivates it

Goal for Lac operon

respond to energy levels in the cell, if low, use lactose as energy source

CAP

Catabolite Activating protein

part of lac operon expression scheme, binds at cis-regulatory sequence.

cAMP

byproduct of glucose metabolism

-an allosteric activator for CAP

• if CAP active and theres no Lac Repressor- there’s high levels of transcription

Bacteriophage Lambda: what is it? what is genome characteristic?

• virus that infects bacteria

• has circular genome

• 48502 bp

• 65 genes

Lysogeny

• inserts into bacterial genome

• proliferates with bacterial dna

• only 1 gene expressed: λ repressor shuts off other genes

• λ repressor high→ Cro: low

lytic cycle

• excises from bacterial genome

• makes bacteriophage particles

• breaks open and kills bacteria

• infects other bacteria

• Cro inactivates λ repressor

• λ repressor: low ; Cro: high

• other proteins expressed that cause DNA damage and kill cell

look at slides 5a: 16-20

bacteriophage λ final thoughts

• bistable molecular switch

• cooperative binding

  • λ repressor forms dimers

  • λ repressor binds to Or2 over Or3

• Cro and λ repressor are mutually exclusive

• premature lysis: phage dies

• lysogeny when bacteria is sick: phage dies

Transcription factor domains

1. one to bind to dna

2. one to interact with other proteins

consensus sequence

OR1: TATCACCGCCAGTGGTA

OR2: TAACACCGTGCGTGTTG

OR3: TATCACCGCAAGGGATA

Levels of control for Eukaryotic Transcription control

1. chromatin accessibility

2. assembly and regulation of protein complexes to initiate transcription

Euchromatin

• open/relaxed

• chromatin that is accessible

heterochromatin

• closed/ condensed

• chromatin that is inaccessible

chromatin accessability

-same dna can be euchromatic or heterochromatic in different cells

involved in assembly and regulation of protein complexes to initiate transcription

1. RNA Polymerase II

2. general transcription factors

3. mediator

4. activators

5. coactivators

• bind close or far away

histone tails

• N-terminal tails of the 4 core histone subunits are conserved.

• directly contact DNA on neighboring nucleosome

histone charge

lysine is (+) charged, binds to (-) charged DNA backbone

Types of histone modification

1. Acetylation of lysines

2. deacetylation of lysines

3. methylation of lysines

Acetylation of lysines

• neutralizes (+) charge

  • histones and DNA interact less tightly

  • dna binding proteins have more access

  • transcriptional activation

deacetylation of lysines

• makes (+) charge accessible

• binds to (-) charged DNA backbone

  • tighter bound

• transcriptional repression

Methylation of lysines

• prevents acetylation

• locks histones in condensed state

• transcriptional repression

DNA methylation

• methylated C: methyl attached to a C

• inherited by daughter cells

Dosage Compensation

when female mammals inactivate one of their two X chromosomes to equalize gene expression with male cells

• X inactivation = random

  • but inherited from mother to daughter cell

• X inactivation= erased during oocyte development

How many genes active on inactive X, and how many inactive on active X?

1

X_ist : What? made of? where it sticks? what it attracts? does?

X_inactive Specific Transcript

• an RNA of ~17kb that do not encode a protein

• Xist mRNA sticks to the X chromosome

• attracts histone methylases

• shuts down entire X chromosome→ Barr body

Insulator Elements

Define genetic regions

Eukaryotic Transcriptional Regulation

• regulates individual genes

• more complex organization

• binding sites may be very far from regulated gene

• intervening DNA loops out

Eukaryotic transcriptional regulation complex organization

• transcription regulators

  • bind DNA

  • + and - regulation

• co-activators

• general transcription factors

• binding/function is often cooperative

Master Regulatory Genes

• powerful genes that induce the development of entire organismal structures

• they are usually transcription factors that activate other genes

• to achieve full effect→ must activate only at a specific time in development and in specific location

Master regulatory genes example 1

Muscle development

MyoD

Gene responsible for change of fibroblasts into muscle cells

• transcription factor

• positively auto regulates itself, locking in a developmental decision

• activates genes necessary for muscle formation

Muscle cell development process

• treat C3H 10T1/2 fibroblast cells with 5-azacytidine causes the cells to spontaneously differentiate into muscle

• in treated cells the genome is less methylated and more genes are expressed

cDNA

complementary DNA

• synthetically made from mRNA template

steps to make cDNA

Necessary Techniques:

1. isolate spliced, poly-adenylated mRNA from cells

2. use viral reverse transcriptase to generate complimentary DNA strand (cDNA)

• most often use a TTTTTT_n primer →because of poly-A tail

• can also use a gene specific primer

• if you generate a second strand you get dsDNA

Nucleic Acid Hybridization

the process of single stranded nucleic molecules come together and form a double stranded structure through complimentary base pairing

1. denaturation

2. reannealing

Denaturation

heating ds nucleic acids will dissociate the strands

reannealing

upon cooling, double stranded nucleic acid will reform

• a labeled ‘probe’ can anneal to the target sequence

  • radioactively labeled

  • fluorescently labeled

MyoD identification process

1. make radioactive cDNA from induced muscle cells

2. hybridize to unlabeled mRNA from fibroblasts

3. DNA/RNA hybrids enzymatically removed

4. remaining cDNAs unique to induced muscle

• housekeeping genes present in both samples→ hybridize and remove using specific enzymes

  • left with 100 muscle specific cDNAs → identify and analyze

test genes left over by adding them individually to fibroblasts and observing function

→ one of the genes was able to convert fibroblasts into muscle

Molecular biology techniques

1. Obtaining DNA

2. DNA analysis

3. DNA amplification

4. Move DNA around

Obtaining DNA

cDNA synthesis

DNA Analysis

• separate by size

• large-scale analysis

• intermediate scale: restriction digests

• nucleotide scale analysis determine the DNA sequence

DNA Amplification

• PCR

• grow DNA in bacteria

Move DNA around

• plasmid DNA

• subcloning

DNA Analysis

• gel made of agarose

  • seaweed polysaccharide

• electrical field applied across gel

• DNA migrates into gel

• separates DNA molecules by size

• visualize by

  • fluorescent DNA dye under UV:

    • dye inserts between stacked DNA basepairs needs many molecules of one size

  • radioactivity:

    • probe only hybridizes to DNA of interest

Large scale:

• can be separated by electrophoresis

• but no sharp bands ( only copies of a single DNA fragment)

• must employ other methods to detect DNA of interest

Steps for separating DNA by size Large scale

1. transfer DNA bands to membrane more stable than gel

2. detect DNA of interest using a complementary DNA probe that is labeled with radioactivity

3. only probe bound to target is stabilized by hydrogen bonds and will remain unbound probe will wash away

4. expose to X-ray film and develop

Steps for separating DNA by size intermediate scale

Restriction Digest

• cut DNA at specific sequences

• cuts inside DNA sequences:

  • endonuclease

• bacterial defense against foreign DNA

• six nucleotide long sites occur ~4kb (4^6)

Steps for separating DNA by size Combining Southern Blot and Restriction Digest

• assume disease with mutation in site 2 for EcoRI: mutant DNA no longer cut by enzyme

• when individual has two mutant chromosomes= disease manifests

• isolate genomic dna

• cut dna

• run on gel

• probe with southern probe

optimized workflow

1. isolate & amplify cDNA

2. fuse to GFP

3. grow DNA in bacteria

4. transcribe into RNA

5. grow embryo

Steps to isolate and amplify cDNA by PCR

1. denaturation/melting ~ 94^oC separates ds DNA molecules

2. Annealing ~54^oC and 65^oC allows primer to ss DNA template

3. Elongation- 72^oC allows DNA polymerase to make new DNA

• repeat ~32 cycles: 1.073 billion correct dsDNA fragments

• product growth:

  • intended- exponentially

  • byproduct- linearly

• after ~ 30 cycles reagents (dNTPs, primers) become limiting

Needs to isolate and amplify cDNA by PCR

• dedicate machine optimized for temperature cycling of small tubes

• DNA polymerase isolated from deep sea vents, ‘stable’ at 95^oC

• buffer with ions and nucleotides

• primers: factory made to desired sequence

• collection of all cDNA from cells making __

plasmids

extra-genomic, circular pieces of DNA

Key elements of a plasmid

• origin of replication (ori)

• antibiotic resistance gne

• multiple cloning site (MCS)

special features: eGFP

Origin of replication (ori)

allows plasmid to be copied

antibiotic resistance gene

allows selective growth of bacteria with plasmid

multiple cloning site (MCS)

DNA with lots of restriction enzymes, insert DNA here

DNA amplification method 3

grow DNA in bacteria

Grow DNA in bacteria

• transform plasmids into bacteria

  • heat-shock treatment

  • electroshock treatment

• select for presence of plasmid (by antibiotic selection)

• grow bacteria, then isolate plasmids

• different plasmids for different purposes

different purposes for plasmids

• make RNA

• make protein

• fuse cDNA to eGFP

• add DNA into genome

in vitro transcription

• make RNA from plasmid

• requires:

  • RNA polymerase

  • ribonucleotides

  • template (our plasmid DNA)

Reduce gene expression

CRISPR and Cas9

• bacterial immune response to bacteriophages

• saves a snippet of phage DNA for later recognition

• destroys phage DNA when reinfected

CRISPR and Cas9

Clustered Regularly InterSpaced Palindromic Repeats and CRISPR Associated